The
monoclonal antibody AR-3 reacts with an
epitope (CAR-3) carried on a high-molecular-weight
glycoprotein associated with
carcinomas of the pancreas, stomach, colon, uterus, and ovary. This study reports the partial purification and characterization of CAR-3-bearing molecule. The
antigen was quantified by a double determinant immunoradiometric assay.
CAR-3 antigen was purified by a three-step procedure, consisting of
perchloric acid extraction, molecular sieving on
Sepharose CL-4B, and affinity chromatography on AR-3
antibodies coupled to
Sepharose 4B. Following this procedure
CAR-3 antigen was purified about 400-fold with a 36% yield. Treatment of the
CAR-3 antigen with 16 mM
metaperiodate or with 1 N NaOH resulted in complete loss of activity. Antigenicity survived enzymatic treatments known to destroy
proteins. The
epitope was found to be carried on a molecule with a molecular weight of greater than 400,000 with a density of 1.45 g/ml, metabolically labeled with [35S]
sulfate, [3H]
glucosamine, and [35S]
methionine. It is concluded that CAR-3
epitope is expressed on a
carbohydrate moiety linked to a sulfo-
mucin-like molecule via an O-glycosidic bond. Cross-competition experiments showed that CAR-3
epitope is strictly related or in close topographic proximity to Lewis(a) and Lewis(b)
antigens. Cross-double determinant immunoradiometric assay experiments indicated that the same
mucin carrying CAR-3 bears also CA 19-9, CA 125, and BW 494
epitopes.